Aim 3: Assess the activity of PNA-loaded PSNPs targeted against miR-122 in vivo
A) Non-destructive optical characterization of in situ synthesis of 23-mer anti-miR122 PNA using reflectometry and Fourier transform analysis to determine the optical thickness of the loaded PSi
4.3 Results and discussion
levels (marker of kidney toxicity) were measured using a commercially available Transaminase- CII kit and Blood Urea Nitrogen-B Test (Wako), respectively. Livers harvested on day 6 were formalin fixed and paraffin embedded and stained with H&E, then evaluated by an experienced veterinary pathologist blinded to the composition of the groups. There was no evidence of liver toxicity observed microscopically.
Mice were fed a standard chow diet ad libitum and had free access to water. All protocols were approved by the Institutional Animal Care and Use Committee of Vanderbilt University and done in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
aggregation [28], potentially contributing to hydrophobic stabilization of the surface coating and stabilization of the drug loading into the PSNP interior pores. At physiologic pH, addition of PEGDB neutralizes negative PSNP surface charge (Scheme 4.1), which is important for systemic delivery applications [112].
Scheme 4.1. PSi-polymer nanocomposite fabrication. 1. Electrostatic assembly of PEGDB on the negatively charged PSNP surface. 2. Purification of nanocomposites by removal of free PEGDB in the supernatant following centrifugation. 3. Buffering the nanoparticle solution to physiologic pH increases polymer self-aggregation, potentially contributing to hydrophobic stabilization of the surface coating and drug loading into the PSNP interior pores. The bottom panel shows surface charge of oxidized PSNPs, PEGDB, and nanocomposites during assembly at pH 5.5, and after buffering pH to 7.4. [Adapted from Advanced Materials, Kelsey R. Beavers et. al., “Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies”, 2016, with permission from John Wiley & Sons, Ltd.]
The ratio of PEGDB polymer to porous silicon was tuned to create a library of nanocomposites with varied degrees of polymer surface functionalization (Figure 4.2). Reactant weight ratios of 1:5, 1:1, 20:1, and 80:1 (PEGDB:PSNP) yield composite particles containing 12, 24, 50, and 60 wt% PEGDB, respectively, as determined by thermal gravimetric analysis (TGA) (Figure 4.2A). Dynamic light scattering (DLS) measurements reveal that as % PEGDB is increased, the PSNP ζ-potential at physiologic pH (7.4) increases and approaches charge neutrality (Figure 4.2B). The average hydrodynamic diameter of nanocomposites with up to 24 wt% PEGDB is the same as that of uncoated PSNPs (220 nm) (Figure 4.2C). Scanning transmission electron microscopy-energy-dispersive X-ray spectrometry (STEM-EDS) analysis of nanocomposites confirms that nitrogen and sulfur signals generated by PEGDB co-localize with the Si matrix at the nano-scale, supporting our hypothesis that electrostatic interactions facilitate PEGDB assembly onto the PSNP surface (Figures 4.2D and 4.3). Spectroscopic evaluation of PSNP colloidal stability reveals that coating of PSNPs with PEGDB minimizes particle aggregation and precipitation in the presence of salt-containing physiologic buffered saline (Figure 4.2E-F). This increased stability can be attributed to PEG’s ability to sterically block surface adsorption of proteins and ions, consequently preventing particle aggregation [117].
Figure 4.2. PEGDB effectively coats PSNPs, neutralizing particle surface charge and imparting colloidal stability within physiologic buffered saline. Characterization of nanocomposite library by A) thermal gravimetric analysis, B) ζ-potential, and C) hydrodynamic size measurements acquired in PBS at pH 7.4. D) STEM-EDS elemental mapping of (top) an uncoated PSNP and (bottom) composite PSNPs partially coated with PEGDB (24 wt%). The increased strength and dispersion of N and S signals in elemental maps of composite particles (bottom) indicates successful PEGDB coating of the PSNP matrix. High-angle annular dark-field images are shown in the left-most panels. Maps of Si, N, and S are indicated in red, teal, and yellow, respectively.
Scale bar = 200 nm. E) Particle aggregation and precipitation out of solution, quantified by monitoring PSNP absorption at 450 nm over time, shows that fully coated 50% PEGDB nanocomposites have increased colloidal stability in PBS. F) Photographs depict PSNP and composite colloidal stability in PBS after 60 min. Black arrow indicates precipitated PSNPs.
[Adapted from Advanced Materials, Kelsey R. Beavers et. al., “Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies”, 2016, with permission from John Wiley & Sons, Ltd.]
Figure 4.3. STEM-EDS Spectra of oxidized PSNPs and 24% Composite PSNPs shown in Figure 4.2D. Cu and Al signals are background from the TEM sample holder. Cl, Na, and P signals are due to the presence of salts from the PBS buffer in which the particles are fabricated. Si is attributed to the porous silicon matrix, and O can be attributed to both the oxide on the PSNP surface as well as within the PEG chain. N and S signals are attributed to PEGDB (see Scheme 4.1 and Figure 4.1 for chemical structure). [Adapted from Advanced Materials, Kelsey R. Beavers et. al., “Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies”, 2016, with permission from John Wiley & Sons, Ltd.]
To assess the impact of PEGDB coating density on PSNP uptake and anti-miRNA activity, composites with the minimum amount of PEGDB necessary to fully shield the porous silicon surface (50 wt%, zeta -3.1 ± 4.0 mV), and composites which were only partially shielded by PEGDB (24 wt%, zeta -8.6 ± 3.0 mV) were selected for further in vitro characterization. Cell internalization and bioactivity was assessed for a PNA designed to inhibit miR-122, a liver-specific miRNA involved in cholesterol biosynthesis. Inhibition of miR-122 is a promising therapeutic approach for reducing viremia in patients infected with Hepatitis C, as well as lowering elevated cholesterol and triglyceride levels due to hypercholesterolemia [118]. In this study, nanocomposites were loaded with anti-miR122 PNA (NH2-ACA AAC ACC ATT GTC ACA CTC CA-cys-COOH) by physically adsorbing PNA within oxidized PSNPs, followed by coating with
PEGDB as described above. The average PNA loading in the nanoparticle formulations was quantified by LCMS to be 34, 22, and 20 nmoles PNA per mg porous silicon (21, 14, and 12 wt%
PNA) for uncoated, partially coated, and fully coated nanocomposites, respectively (Figure 4.4 and Table 4.2). To our knowledge, this is the highest reported PNA loading in any nanoparticle system, and is 60x higher than what has been reported for anti-miRNA PNA loading in PLGA nanoparticles [69], highlighting an important advantage of highly porous PSNPs that do not require emulsion fabrication/loading approaches.
Figure 4.4. LCMS chromatographs used to evaluate PNA loading in PSNPs, showing how the polymer component of the composite was separated from PNA for drug loading quantification.
The highlighted peaks were confirmed to be pure anti-miR122 PNA by mass spectrometry.
[Adapted from Advanced Materials, Kelsey R. Beavers et. al., “Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies”, 2016, with permission from John Wiley & Sons, Ltd.]
Table 4.2. PSNP PNA loading calculated from LCMS.
Sample PNA/PSNP (wt%) PNA (nmoles mg-
1)
Empty Comp 0 0.0
PNA-Loaded Full Composite 12 19.8
PNA-Loaded Partial Composite 14 21.9
PNA-Loaded Uncoated PSNPs 21 34.3
Cellular uptake and miR-122 inhibition studies were performed in vitro using Huh7 human liver cancer cells (Figure 4.5). Cells were treated for 24 h in Dubelcco’s modified eagle medium (DMEM, Gibco Cell Culture, Carlsbad, CA) supplemented with 10% fetal bovine serum, at a 2µM dose of PNA. Following treatment, uptake of fluorescently-labeled PNA was quantified by flow cytometry and imaged by confocal microscopy. PNA encapsulated in uncoated PSNPs is ~50x more efficiently internalized than free PNA (Figure 4.5A). Additionally, PNA uptake decreases proportionately with increasing wt% PEGDB (Figure 4.5A and 4.5B). This is likely due to PEG shielding on the outer surface of the composite [119]. Importantly, the extent of cytosolic PNA delivery increased with increasing PEGDB content, as quantified by co-localization analysis of PNA and lysosomes stained with LysoTracker®, 24 h after treatment (Figure 4.5C). This enhanced cytosolic PNA delivery can be attributed to both full and partial PEGDB nanocomposites possessing pH-dependent, membrane disruptive activity in a relevant endo-lysosomal range, whereas uncoated PSNPs do not (Figure 4.5D).
Figure 4.5. Coating of PSNPs with PEGDB decreases PNA uptake but increases both endosome escape potential and anti-miRNA activity relative to uncoated PSNPs in Huh7 human hepatocellular carcinoma cells. PEGDB functionalization decreases cellular uptake, as characterized by A) flow cytometry and B) confocal microscopy, 24 h after treatment with Alexa Fluor 488-labeled anti-miR122 PNA at a 2 × 10–6 M PNA dose. Top scale bar = 100 μm, bottom scale bar = 10 μm. C) PEGDB functionalization increases PNA cytosolic delivery, as shown by
colocalization analysis of Alexa Fluor 488-labeled PNA with LysoTracker at 24 h after treatment with 2 × 10–6 M PNA. Endosomal entrapment was quantified by calculating the Manders' overlap coefficients for green and red pixels, shown at the right as means ± SEM (n ≥ 3 separate images).
Increased cytosolic delivery observed for composite particles is due to D) the pH dependent membrane disruptive function (grey arrow) of PEGDB, as determined by a hemolysis assay.
Composites did not disrupt erythrocyte membranes at pH 7.4, but produced robust hemolysis at pH 6.2, which is representative of late endosomes. E) A firefly luciferase assay reveals that all PSNP treatments are non-toxic at a 2 × 10–6 M PNA dose, in contrast to the gold-standard of a 2′OMe modified RNA delivered using a commercial cationic transfection reagent (AMO+Fugene6). F) Therapeutic anti-miR122 activity increases with increasing PEGDB polymer functionalization (based on Renilla luciferase readout tied directly to miR-122 inhibition) 24 h after treatment, when compared to free, unencapsulated PNA and the control, 2′OMe AMO (p <
0.05 when compared to *Free PNA or PSNP, **AMO+Fugene6, and ***Partial Comp). [Adapted from Advanced Materials, Kelsey R. Beavers et. al., “Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies”, 2016, with permission from John Wiley & Sons, Ltd.]
Anti-miR122 activity was quantified using Huh7 cells stably transfected with a Renilla luciferase sensor for endogenous miR-122 [115]. Inhibition of miR-122 in these cells causes an increase in luciferase signal. Both anti-miRNA activity and cytotoxicity were benchmarked against the anti-miRNA oligonucleotide (AMO) agent used in development and validation of this luciferase reporter cell line: 2’OMe PS modified RNA delivered with the cationic commercial transfection reagent, FuGENE® 6. All PSNP treatments cause significantly less cytotoxicity than FuGENE® 6 (Figure 4.5E), which is too toxic (and colloidally instable) for in vivo translation.
Although cell uptake was reduced (Figure 4.5A), anti-miR122 activity was 6-fold and 10-fold greater than uncoated PSNPs for the partial and fully coated nanocomposites, respectively (Figure 4.5F). Furthermore, fully coated nanocomposite PSNPs demonstrate 2.3 fold higher miR-122 inhibition relative to the AMO standard. Taken together, these data suggest that fully coated composites are non-toxic and have potent anti-miRNA activity due to increased delivery of PNA to the cytosol, where miR-122 is located.
To evaluate whether nanocomposites improve the blood circulation half-life and miRNA inhibitory bioactivity of PNA in vivo, CD-1 mice (10 weeks of age, Charles River) were injected intravenously via the tail vein with 1 mg kg-1 cy5-labeled anti-miR122 PNA (free, loaded into uncoated PSNPS or fully coated nanocomposites) (Figure 4.6). Blood samples were collected 5, 10, 40, and 80 minutes after injection, and circulation half-life was determined based on the quantity of PNA in the plasma collected at each time point (Figure 4.6A). Uncoated PSNPs extended the circulation half-life of free PNA from <1 min to ~30min, and addition of the PEGDB coating to PSNPs more than doubled the half-life to nearly 70 min. As a result of increased circulation time, encapsulation of PNA anti-miR122 in nanocomposites increased its bioavailability by 73x, as quantified by the area under the curve (AUC).
The organ biodistribution of PNA and the PSNP carrier were determined by excising the heart, lungs, liver, spleen, and kidneys 160 min after injection. Investigations into novel nucleic acid delivery systems typically track the fluorescently-labeled nucleic acid without tracking the carrier system. An advantage of using porous silicon nanocarriers is that their biodistribution can be tracked label-free using inductively coupled plasma-optical emission spectroscopy (ICP-OES).
Figure 4.6B compares the biodistribution of fluorescently labeled PNA cargo with that of Si from the porous silicon nanocarriers. The biodistribution of Si is similar to that of the PNA for both uncoated and composite nanoparticles, suggesting that the PNA cargo may remain stably associated with the porous silicon carrier in the circulation and during initial tissue biodistribution.
It is well established that nanoparticles larger than 200 nm in size are preferentially sequestered in the liver sinusoidal endothelium [120]. Due to their size, PSNPs and nanocomposites increased PNA delivery to the liver (where hepatocytes containing the target miR-122 are located) by ~16%, and reduced the amount of PNA in the kidneys by ~33% when compared with free PNA (Figure
4.6B). ICP-OES analysis reveals a ~20% increase in Si in the spleen of composite particles when compared to uncoated particles. The spleen, like the liver, is known to play a primary role in nanoparticle clearance. Additionally, PEGDB coated nanoparticles display 12% less Si accumulation in lungs compared with uncoated PSNPs 160 min after injection, and 29% less lung bioavailability over 24 h (Figures 4.6 and 4.7), corroborating in vitro data (Figure 4.2 E-F) that PEGDB reduces flocculation which causes accumulation in the lungs [121]. A final observation is that while ~20% of all PNA is found in the kidneys, no more than 6% of all measured Si is detected in the kidneys by ICP-OES. Free PNA pharmacokinetics data suggests that the kidney is a preferential route of clearance, which is anticipated based on the small size of free PNA, below the renal cutoff. The difference in PNA and Si content suggests that a portion of the PNA cargo released from the PSNP carriers in circulation due to the noncovalent PNA loading mechanism.
We next tested whether PSNP-polymer nanocomposites improve PNA bioactivity (miR- 122 inhibition) in the liver. Female C57BL/6J mice (12 weeks of age, Jackson Laboratories) were treated with either saline, free anti-miR122 PNA (5 mg kg-1 PNA), PNA loaded into composite PSNPs (5 mg kg-1 PNA, 42 mg kg-1 PSNP composite), or an empty composite vehicle control (42 mg kg-1 PSNP composite). Importantly, the uncoated nanoparticles demonstrated poor colloidal stability in physiologic solutions (Figure 4.2E-F). We were unable to safely inject uncoated PSNPs intravenously at 5 mg kg-1 PNA dose for this study due to acute mortality. The acute toxicity for the 5 mg kg-1 uncoated treatment group could be due to particle aggregation resulting in blockage of pulmonary capillaries. This result highlights the importance of the colloidal stabilization of PSNPs by PEGDB, which significantly reduced particle accumulation in the lungs (Figures 4.6C and 4.7).
Figure 4.6. PSNP-polymer nanocomposites increase PNA blood circulation half-life, bioavailability, and anti-miRNA activity in vivo. A) Blood pharmacokinetics curves generated using cy5-labeled PNA show that PSNP composites increase circulation half-life of PNA when delivered I.V. through the tail vein of mice at a 1 mg kg−1dose (n = 8 per group for 0–20 min, n = 5 per group for 40–80 min). B) In vivo biodistribution of cy5-PNA cargo and Si from the PSNP carriers was analyzed by fluorescent imaging and ICP-OES, respectively. PNA and Si organ distributions 160 min after injection show that PSNPs increase PNA accumulation in the liver and decrease uptake in the kidneys. C) Quantification of bioavailability in blood, liver, and lungs demonstrates that PEGDB functionalization improves blood circulation stability and decreases
accumulation of particles in the lungs. D–H) In vivo miR-122 inhibition studies following injection of a 5 mg kg–1 dose of PNA, every other day for 6 d (n = 6 mice per group). D) Livers were evaluated by an experienced veterinary pathologist blinded to the composition of the groups, who noted no evidence of liver toxicity observed microscopically (representative image shown,n = 6 mice per group). E) Nanocomposite delivery of anti-miR122 PNA D) significantly inhibits miR- 122 and F) modulates the expression of miR-122 direct target genes, Aldoa and Gys1, in addition to the indirect target gene MTTP (Grey line indicates saline control). Cholesterol measurements on G) HDL and H) LDL fractions separated by fast protein liquid chromatography (FPLC) from plasma collected on day 6 reveals decreased cholesterol in HDL following treatment with nanocomposites loaded with anti-miR122 PNA. (*, **, and *** indicate p < 0.05 when compared to free PNA, PSNPs, and empty vehicle control, respectively). [Adapted from Advanced Materials, Kelsey R. Beavers et. al., “Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies”, 2016, with permission from John Wiley & Sons.]
Figure 4.7. Biodistribution of cy5-PNA in the major organs as a function of time, following a single intravenous injection of A) 1 mg kg-1 free PNA, B) 1 mg kg-1 PNA loaded into uncoated PSNPs, and C) 1 mg kg-1 PNA loaded into composite nanoparticles (n > 3 mice/group). A non- linear, one-phase exponential decay model was used to generate pharmacokinetics curves for each data set (average curves shown as solid lines in charts A-C). D) PNA bioavailability was then characterized by calculating the area under the curves from 0-24 h. (* and ** indicate p<0.05 when compared to free PNA and PSNPs, respectively). [Adapted from Advanced Materials, Kelsey R.
Beavers et. al., “Porous Silicon and Polymer Nanocomposites for Delivery of Peptide Nucleic Acids as Anti-MicroRNA Therapies”, 2016, with permission from John Wiley & Sons, Ltd.]
Mice were injected intravenously into the tail vein every other day for 6 days (3 injections), then sacrificed, and their livers were harvested for mRNA, and miRNA analysis (Figure 4.6 D- F). There were no overt signs of toxicity for the composite nanoparticles, and levels of blood urea nitrogen (BUN) and alanine aminotransferase (ALT) measured on day 6 serum samples collected at the time of mouse sacrifice suggested that treatment did not cause kidney or liver toxicity relative to control treatment groups (Figures 4.6D and 4.8). Real-time PCR for miR-122 expression shows that composite PSNPs inhibited miR-122 by 46% relative to the empty vehicle control (Figure 4.6E). Additionally, treatment with nanocomposites caused an ~50% increase in expression of both Aldolase A (Aldoa) and Glycogen Synthase 1 (Gys1), validated miR-122 gene targets that encode for proteins which degrade cholesterol and synthesize glycogen, respectively (Figure 4.6F). Furthermore, expression of Microsomal Triglyceride Transfer Protein (MTTP), an indirect target of miR-122 known to be down-regulated upon miR-122 inhibition, was decreased by 36%
relative to the vehicle control (Figure 4.6F) [122]. Finally, the functional impact of miR-122 inhibition was assessed by analyzing the cholesterol content in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL) serum fractions collected from mice on day 6 (Figure 4.6G- H). Consistent with the known function of miR-122 [118], inhibition of miR-122 using PNA- loaded nanocomposites caused an ~20% decrease in HDL cholesterol. This is, to our knowledge, the first demonstration of in vivo PNA-mediated miR-122 inhibition, and the nanocomposite PNA delivery technology exhibits miR-122 inhibition at 16x lower dose than RNA-based anti-miR122 antagomirs [42], and 2.5x lower dose than a 2-O-methoxyethyl phosphorothioate AMOs [118].
Systemic and intracellular pharmacokinetics limitations are the biggest challenges facing PNA-based therapeutics. Our in vitro and in vivo results confirm that free PNA suffers both from poor cellular internalization, poor systemic pharmacokinetics, and lack of intracellular
bioavailability. Our in vitro data support the importance of overcoming intracellular delivery barriers. Despite a significantly higher level of cell uptake, the uncoated PSNP carriers produced significantly lower miRNA inhibition than composite particles with active endosomal escape capacity (Figure 4.4). Furthermore, the nanocomposite showed superior blood circulation time and systemic bioavailability (Figure 4.6) due to particle colloidal stabilization with PEG. Finally, PEGDB polymer coating enabled I.V. delivery of a 5x higher PNA dose than uncoated PSNPs. At this PNA dose (5 mg kg-1), composite particles successfully inhibited miR-122 in the liver, de- repressed the miR-122 target gene, Aldoa, and lowered plasma cholesterol levels. Thus, the nanocomposite design was strategically crafted to overcome both the key systemic and intracellular delivery barriers facing PNA.